Each of the papers in this Arabidopsis Research Roundup involves the response to different stimuli. Giles Johnson at Manchester provides an audio description of work that has discovered a novel mechanism of cold sensing whilst Gordon Simpson and John Brown from Dundee are contributors to work that has interrogated the sugar signaling pathway. Finally is a study from Warwick that has identified novel loci involved in ABA signaling and seed vigour.

From http://www.plantphysiol.org/content/early/2016/07/20/pp.16.00852.long

Giles Johnson (Manchester) is the corresponding author on this UK-German collaboration that looks at the mechanisms by which plants sense the low temperatures that cause significant phenotypic changes. GC-MS showed that fumarate is a key component in the cold tolerance response and that the activity of the FUM2 enzyme is responsible for accumulation of fumaric acid. Plants that lack FUM2 activity show significant alteration in gene expression and metabolite profile following a cold treatment and in particularly are unable to acclimate photosynthesis at lower temperatures. Therefore this study introduces a novel component of the temperature sensing apparatus, which might have broad significance for attempts to develop crops with an improved cold response.

Giles kindly provides an audio description of this work, which includes an overview into cold acclimation of photosynthesis. This includes an excellent ‘stress-ball’ analogy! (Apologies for pen-clicks :/).

From http://www.plantcell.org/content/early/2016/07/19/tpc.16.00301.abstract

Gordon Simpson and John Brown (James Hutton Institute) are contributors to this Portuguese-led study that investigates the role of the SR45 splicing factor in sugar signaling. In sr45-1 mutants they show that glucose-feeding causes increased levels of the energy-sensing SNF1-Related Protein Kinase 1 (SnRK1) yet without increasing its gene expression. Concomitantly the hypersensitivity of sr45-1 mutants is rescued in plants with reduced levels of SnRK1. The authors discovered that the mechanistic link between these genes involves SR45-1 regulating the alternative splicing of the 5PTase13 gene, which encodes an inositol polyphosphate 5-phosphatase that interacts with SnRK1 in vivo. In wildtype plants 5PTase13 modulates proteasomal-mediated degradation of SnRK1 and therefore a perturbation of this process in sr45-1 explains this defect in sugar-sensing.

Bill Finch-Savage is the corresponding author on this study from the Warwick University that uses Brassica oleracea natural variation to identify novel loci involved in seed vigour. The discovered QTL was termed Speed of Germination (SOG1) and contained two genes, BoLCVIG2, a homologue of the alternative-splicing regulator (AtPTB1) and BoLCVIG1, which has unknown function. Transfer of these alleles into Arabdopsis causes alterations in seed germination, which is also observed in mutants of the equivalent Arabidopsis genes (At3g01060, At3g01150). Furthermore an additional discovered QTL encodes the Reduced ABscisic Acid 1 (RABA1) gene, which influences ABA content and seed vigour. Therefore this mapping strategy has discovered three genes that promote seed vigour resulting from alterations in ABA content and sensitivity.

The annual Arabidopsis conference was held in GyeongJu in South Korea this year. I was fortunate to go, thanks for some extra funding from the Gatsby Foundation and GARNet.

Going to the conference I of course looked forward to my own supervisor Lars Østergaard’s talk. But I also had some other sessions and speakers I was really excited about. And they did not disappoint. Sabrina Sabatini wonderfully explained her elegant model of auxin and cytokinin distribution in the root, Dolf Weijers showed some stunning images of the Arabidopsis embryo which were only rivalled by Minako Ueda with her live-cell imaging of cell divisions, microtubule arrangement and mitochondria in the zygote. There was also Annika Weimer from the Bergmann lab who told the story of how formative divisions occur in the root and the stomata, followed by a great description of the sepal cellular arrangements by Adrienne Roeder. She outlined the effect of ATML1 levels on cells division versus endoreduplication.

Tom Beeckman’s talk was also great where he presented his Science paper, published in January, on a cyclical auxin signal coupled to cell death which determines lateral root formation. I had read his paper and discussed it in a journal club but it was brilliant to hear the story told by the person most excited and motivated about it! One of the most important things I learned from these sessions was not to dismiss Domains of Unknown Function (DUFs). Hunting down a DUF, Dolf Weijers and his lab found a protein beautifully localized in the corner of cells, playing a role in determining the cell division plane. And who wouldn’t want to name a protein!?

I also want to highlight two talks from the transporter session, a topic not directly relevant to what I do. Yi-Fang Tsay neatly described her research on nitrogen transporters. She identified one that was involved in transporting N from old to new plant parts in times of low N (NRT1.7) and others that transported nitrogen in high N conditions (NRT1.11 and NRT1.12). The other talk, by José A Féijo, showed that transport in the plant can be a lot more complex, with a large network of genes involved in calcium transport in the pollen tube.

But for me the best talk came from a very relaxed and confident John Bowman in the session on shoot development. His theory that leaves are modified shoots has led him to attempt to modify leaves in a way that they revert back to the indeterminate growth of shoots. And he has succeeded, producing Arabidopsis leaves that are crinkled, a little like kale, and grow indeterminately. His talk was delivered in a very clear manner and kept me fascinated all the way through, even though he was the last of his session. And even though it was my favourite talk, it was one I took the fewest notes on and I believe that is because I was so captivated by his story that I just sat back and listened and simply forgot about notetaking!And there is one more thing I really got to appreciate at this, my first large conference: that the talks are just one part of the event. The few hours you get around the talks; during coffee and lunch times, dinner, poster sessions and drinks, are the times that probably matter just as much. They give you the chance to talk to the people you have just seen present. They give you the chance to meet people you admire and talk to them on a first name basis. They allow you to meet scientists from all over the world, from all career stages. They make you realise that everyone else, same as you, is there to have a great time talking science and experiencing a different country. And most of all, it makes you appreciate that in the end, no matter what stage of your career you are, you are part of a community that is interested in a common topic, driven by curiosity and a thirst for knowledge.

There are six papers in this weeks Arabidopsis Research Roundup. Two of these include research on the stomatal patterning gene TMM. Firstly a White Rose consortium investigates the ancestral basis of stomatal patterning, whilst a Glasgow-based study investigates the relationship between patterning and the dynamics of guard cell opening. The GARNet committee is represented by work from Cardiff that looks at the relationship between seed size and shoot branching and also from Cambridge in research that studies meiotic recombination in genomic regions important for pathogen defense. Finally are two studies that look into aspects of root and shoot patterning and include co-authors from CPIB in Nottingham or the John Innes Centre.

This study is a collaboration between labs in Sheffield and Leeds, led by Andrew Fleming (Sheffield). They investigate the role that the signalling module comprised of Epidermal Patterning Factors (EPFs), ERECTA and TMM play during the evolution of stomatal patterning. This module is known to play an important role in Arabidopsis and in this study the authors show that the moss Physcomitrella patens contains homologs of each of the genes and that they perform the same function. When P.paten versions of these genes are transferred to equivalent Arabidopsis mutants they show conserved function demonstrating that this module is an example of an ancestral patterning system.

Mike Blatt and Anna Amtmann (University of Glasgow) are the co-supervisors for this study into the relationshop between ion transport in stomatal guard cells and their physical positioning within a leaf. They used a genetic approach to assess the effect of stomatal clustering, showing that too many mouths (tmm) mutant plants have reduced stomatal movements associated with alterations in K+ channel gating and coincident with a surprising reduction in the level of K+ ions in guard cells. These results underline the importance of stomatal spacing in this process but do not provide a full explanation into the alteration in K+ ion dynamics.

This research comes from the lab of GARNet PI Jim Murray (Cardiff) and investigates cell proliferation and growth within a developing seed. They previously have shown that targeting of D-type cyclin CYCD7;1 to the central cell and early endosperm can trigger nuclear divisions and ovule abortion, which leads to a smaller number of larger seed. In this study they show that development of larger seed in transgenic plants is influenced by the architecture of the mother, as plants with increased side branches, caused by pruning of the main stem, do not generate this phenotype. This is indicative of a close relationship between the amount of resources allocated to different parts of the plant and that a transgenic effect was altered by a different plant morphology. This should provide an important insight into future work that aims to define the effect of any particular transgenic alteration.

GARNet advisory board member Ian Henderson (Cambridge) is the corresponding author of this study that involves contributions from the UK, US, Poland and France. They investigate genomic regions that show increased meiotic recombination, which is predicted to occur coincident with genes involved in pathogen defence given their requirement to adapt to new external challenges. This study focuses on NBS-LRR domain proteins that tend to physically cluster in the Arabidopsis genome. Interesting they discovered both hot and coldspots for meiotic recombination that associate with NBS-LRR clusters, the later often correlating with structural heterozygosity. In a more detailed dissection of 1000 crossovers in the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R hotspot, they discovered higher recombination frequencies associating with known sequence motifs important for the pathogen response, which were influenced by ecotype-specific factors. Ultimately the authors note that there is a complex relationship between regions of meiotic recombination, structural heterozygosity and the evolutionary pressures that occurs with host-pathogen relationships.

Malcolm Bennett (CPIB) is the sole UK-based co-author on this study led by Belgian collaborators and investigates the role of reactive oxygen species (ROS) in auxin-regulated lateral root (LR) formation. They show that ROS can reactivate LR primordia and pre-branch sites, resulting in increased LR numbers. This occurs in both wildtype and in auxin mutants that have reduced numbers due to changes in auxin-mediated cell wall remodeling. ROS is deposited in the apoplast of emerging LR cells in a pattern that is coincident with the expression of the RESPIRATORY BURST OXIDASE HOMOLOGS (RBOH) genes. Concomitantly the altered expression of RBOH was shown to affect the development and emergence of LRs. This adds a further level of complexity to the current understanding of the signaling factors that converge to facilitate LR growth.

This Chinese-led study includes Robert Sablowski (JIC) as a co-author and studies the factors that influence the development of axillary meristems. They use innovative live imaging to show that SHOOT MERISTEMLESS (STM) is continuously expressed and that this dependent on a leaf axil auxin minimum. Once STM expression is lost then the axil is unable to form a meristem even if STM is switched back later in development, indicating that cells undergo an irreversible developmental commitment. The expression domain of STM is under cell-type specific control of REVOLUTA (REV) DNA binding. Overall this study demonstrates that meristematic competence and initiation is dependent on differing levels of the key regulator STM.

This summer, from 29th June to 3th July, I had the honour to attend and give a talk about my research at the International Conference of Arabidopsis Research 2016 at Gyeongju, Korea. The venue for the conference, HICO, was in the green mountains next to a peaceful reservoir. All this gave the conference a romantic and scenario touch.

The conference started promptly with the keynote from Prof Jen Sheen of Harvard University. She gave three nice examples of how her group looking for missing links in signalling pathways using genetic tools. It was particular useful to know that an ultrasensitive Calcium sensor is available out there. In the rest of the whole conferences, there are more talks with these highlights of technology, giving me useful updates of the cutting edge techniques in science research today.

In the five days of conference progression, I enjoyed Youngsook Lee’s nice overview of ABC transporters, Annika Weimer’s fascinating claim that cyclin dependant protein kinase controls the expression of meristemoid lineage gene SPCH, Doris Wagner’s outstanding epigenetic approach to increase plant water use efficiency by the brm gene.

Most importantly, it was the first time for me to give a talk on such a big platform and to such a big audience. The talk went very well and I received some constructive questions and suggestions, as well as some toughening grilling. Throughout the process I got to know some very talented researchers from all over the world, giving me information both on scientific projects and other relevant information.

This leads to the social part of the conference. Meeting new people is always one of my favourite part of academic conferences. It is always encouraging to know there are so many bright minds and great people out there, doing great things and willing to support each other in the community. I myself managed to reconnect to a couple of old friends in America, and to know a few more friends from UK and all over the world. Besides, there was one fresh experience this time. As the conference was held in Asia, I was able to talk to many fellow Chinese people, have some great chats and get a better idea of the current picture of plant science in China. Some of them, due to the language barrier or other issues, did not make international contacts despite high quality research outputs. This is truly a shame for the whole community and hope things can change a little bit in the future.

If there were some lessons to be learnt, I certainly hope, in the future, before attending such a big meeting, I would sit down and go through all the poster abstracts and prioritise the ones I would like to see most. When there were over 500 posters in the same room, there was hardly enough time to see them all and discuss with their authors properly.

In conclusion, I thoroughly enjoyed the meeting and grateful to GARNet and Gatsby foundation to support me to go there. It was great to be immersed in such an international ambience and absorb knowledges and ideas from different aspects. And as I said in my talk, science should be all about open and collaboration, and carry this globalism spirit forward whatsoever.

As part of the Career Chat Workshop at the meeting, Bethany Huot from the Michigan State University took notes from a general discussion with Michael Gonzalez who works as a Plant Biology Consultant. Her summary of this discussion was published on the excellent Plantae resource but given its unusual subject matter I thought it would be good to re-post here on the GARNet blog.

At the 2016 Plant Biology conference in Austin, Texas, we had the opportunity to sit down with Michael Gonzalez to get tips on pursuing a career as a Plant Biology Consultant. Here are some of the tips and suggestions Michael shared with us.

Getting Started:

The first step towards establishing yourself as an independent consultant is to identify what skills you have to offer and how you can market those skills. Some areas of expertise that Michael has seen used successfully in this way are computational, educational or in plant breeding! As a self-employed person, some basic skills you will need to develop are time and money management, as these are the most critical factors for your short and long term success. Some specifics on these and other necessary skills are detailed below.

Building your clientele:

The first step towards making your new business successful is to start building your clientele. The key here is Networking and to make the most of each conversation, you will need to be prepared. Michael suggests writing down a mission statement based on what you do well and practicing it as a sales pitch. This way you will be sure to hit on every key element of what you have to offer during any particular conversation. One thing to keep in mind is to provide enough information to gain interest, but not so much that you enable your potential client to take your ideas and run with them on their own.

Another critical element is developing a Marketing strategy. To maximize your networking opportunities, you will want to increase your visibility. In addition to the traditional business cards, Michael also recommends using online tools, like setting up your own website and blogging on sites frequented by potential clients. Bare in mind however that the best marketing tool is face-to-face interaction! Michael’s advice for this is to be prepared with that sales pitch and, above all, sound professional. Michael repeated this tip multiple times, emphasizing that the extent to which you are prepared and sound professional will go a long way towards closing the deal. The best way to prepare for each meeting is to not only rehearse what you have to offer, but to also research the person with whom you are interacting, what it is they do and what specifically you have to offer them in their research. Michael says potential clients are looking for someone who can come in and get the job done, not another graduate student they will need to manage. [Also remember that any random conversation could lead to something in the future so always maintain your professionalism- GARNet note]

Finally, while having clients is obviously critical to establishing a solid business, you will want to be selective about the clients with whom you agree to work. Entering into contracts with the wrong people can be more trouble than its worth, and may potentially put your business at risk. Remember that building a solid reputation is an essential element of establishing a sustainable business, and the people with whom you do business reflect on you as well.

Negotiating your contract:

Two of the main tips here are to develop your negotiation skills and to get your contract in writing. No matter how nice or honest a client appears, you do not want to take the risk of someone “forgetting” the deal to which you both agreed prior to you doing the work.

This brings us to the delicate issue of setting a consulting fee. Of course you do not want to over price yourself, but you also need to be able to pay your bills and have some money left over for savings and spending. Michael shared with us that one of the mistakes he made as a new consultant was failing to account for having to pay his own taxes, healthcare and retirement savings. In general, he advised estimating ~30% above what your salary would be if you were employed by a company. If you aren’t sure what average salaries are for people employed in your area of expertise, you can find out by using online sources, such as PayScale, Glassdoor, Monster and many others. When doing this, use more than one source and be mindful of the job location to be sure the number is reasonably accurate.

The key here is to be sure how much your time is worth before entering into a contract fee negotiation. Michael also advised to keep in mind who your client is and adjust up or down based on what they can afford. It is also fine to ask for more and negotiate down, but know your lower limit. Of course, with each kind of client there come different challenges. One specific example Michael provided was that when working for academic clients, one issue in getting your desired fee could be that it is higher than the salary of the PI who is hiring you, which would be difficult for them to justify. In this case he suggests compensating for the lower salary by asking for additional money to cover travel or equipment, which will be tax deductible business expenditures.

Determining which jobs to take and which to decline is a balancing act between the amount of time and effort you will need and want to devote to a project and how much money they want to pay you to get the job done. Another thing to consider in negotiating your contract is whether to set your fee based on the project or by the hour. If by the project, you should have a reasonable estimation of how much time it will take you to complete so you do not end up shorting yourself.

Finally, Michael also advised keeping your long-term goals in mind when setting your contract parameters. While you may be excited to be self-employed, you may not want to remain so forever. In the event you do decide to return to work for someone else, whether in academia or industry, you will want a publication record – including posters, papers and patents – to which you contributed. Along this line, when you agree to do work for someone at a company or a University, be sure you are clear on what you are agreeing to give them. Michael cautioned us to verify the contract distinguishes between using your intellectual property for someone and developing intellectual property for them. The difference is in who owns your work when you are done! One thing to avoid is accepting equipment, such as a work computer or email address, from a client as any work done using this equipment, including emails, will belong to them. Michael again emphasized the need to know what you want and to what you are agreeing when negotiating your contracts.

Formalizing your business:

Be sure to know the different types of business before committing to one. Michael recommends going with a LLC (limited liability company) over a sole proprietor option. The main reason? Going with sole proprietor makes all of your personal assets vulnerable if a client decides you did not fulfill your contract obligation and sues you. With an LLC, you open a separate bank account to use for all business expenses, and only the money and assets associated with this account are at risk in a lawsuit. (For more information on this, click here.) Michael also recommends using Legal Zoom, as it is a user friendly, affordable tool for getting the appropriate paperwork together for your business.

In Summary:

Overall, Michael provided us with some valuable advice regarding a career option many of us may have overlooked or been too intimidated to consider. Even if you prefer to work for someone else, these tips can help you be more attractive as an applicant. Thanks, Michael, for taking your time to share your expertise.

On the final morning of the recent ICAR2016 meeting attendance was rather low, seemingly after a heavy karaoke-filled night! However those that got out of bed had to pleasure of attending the excellent ‘Plant Biotechnology’ session, which provided great insights into the process of developing and bringing plant products to the market.

However for me it was a more ‘conventional’ presentation that was a real eye-opener. Li Ling from Iowa State University introduced the QQS gene that is an orphan gene only present in Arabidopsis. This small loci has no previously characterised domains and was identified during expression analysis of genes that are upregulated in a strach synthase mutant. Although it is small and mysterious, the QQS gene appears to play a remarkably role in the control of plant carbon-nitrogen balance. Arabidopsis plants that overexpress QQS show a significant decrease in leaf starch content and an associated increase in overall leaf protein content. This occurs without any phenotypic change or yield penalty.

Li Ling subsequently overexpressed QQS in soybean and also showed, even though plants were again morphologically identical to wildtype, an increase in seed protein and only a slight decline in seed oil content. Remarkably this is occuring in plants that have been previously ‘seen’ the QQS gene and this effect occurs independent of growth location (greenhouse, field) or soil nitrogen content. Similar results have been also found when QQS is overexpressed in maize and rice.

In more recent experiments Ling and co-workers discovered that QQS acts upstream of the AtNF-YC4 gene in Arabidopsis and that overexpression of AtNF-YC4 also causes an increase in protein content and a decrease in starch. Soybean, maize and rice each contain orthologs of AtNF-YC4, demonstrating that the signalling module is preserved in these distantly related crops. Li mentioned that attempts to modify the expression NF-YC4 in crop plants using gene-editing techniques are at an advanced stage, which will hopefully lead to the generation of transgene-free plants that have increased protein content. Furthermore Li reported that the QQS:NF-Y4 module influences multiple aspects of the response to pathogens, work which will be published soon and further demonstrates the pleiotropic nature of QQS function.

From http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664325/

Overall the QQS gene has remarkably potential for use in experiments that aim to generate plants with altered C/N balance. It seems that even though Arabidopsis, soybean, maize and rice are evolutionarily divergent, they each retain the ability to respond to the QQS protein via the NF-YC4 signaling module.

As the authors state, the yield-altering effects of this response pathway have broad societal importance and hopefully can be used in the generation of crops with different nutritional attributes.

On a smaller scale, this work with an orphan gene should also offer encouragement to researchers who have identified a gene with ‘no known function’ from their particular mutant screen! If you persevere, you never know where it might lead!

After a conference break the Arabidopsis Research Roundup returns with an outstanding selection of papers from UK (and mostly Scotland-based) researchers. Firstly Levi Yant provides an audio description of work that has identified important loci for adaption to harsh environments. Secondly John Doonan leads a multi-national group investigating the role of eiF4A phosphorylation within proliferating cells. Next two Scottish-based studies both investigate aspects of light signalling on different scales: a Glasgow-based consortium dissects the UVR8 signaling module while the role of phytochrome on global carbon allocation is studied by Karen Halliday’s group in Edinburgh. The final paper also involves significant Scottish involvement with Piers Hemsley at Dundee together with Simon Turner at Manchester investigating the role of s-acylation in the activity of the cellulose synthase complex.

New investigator at the John Innes Centre, Levi Yant, is the corresponding author on this study that also includes contributions from the labs of Kristen Bomblies and current GARNet Chairman David Salt. This investigation uses GWAS techniques to identify loci in Arabidopsis Arenosa that are important for growth on serpentine barrens, which are characterised by drought, mineral paucity and high levels of heavy metals. They showed that polygenic multi-trait genomic locations are important for serpentine adaptation. The authors reassessed previous independent datasets and showed that 11 loci have been identified across these studies and are therefore good candidates as drivers of convergent evolution. This study provides evidence that certain A.arenosa alleles have been introgressed from A.lyrata and that these may facilitate adaptation to a multi-hazard environment.

Levi kindly provides a short audio description of this work, that also touches on ionomics and data reuse!

John Doonan (Aberystwyth) is the leader of this wide collaboration of UK, US, Czech, Greek and Chinese researchers that investigate the interaction of the eIF4A RNA helicase with cyclin-dependent protein kinase A (CDKA). This interaction only occurs in proliferating cells where CDKA acts by phosphorylating specific amino acids on eIF4A. Throughout in vivo and in vitro experiments using phospho-null and phosphor-mimetic version of eIF4A, the authors show that phosphorylation acts to downregulate eIF4A activity, subsequently altering the efficacy of translation.

This exclusively University of Glasgow study is led by John Christie and Gareth Jenkins. Dimeric UVR8 is a UV photoreceptor that after UV-B interaction dissociates into monomers, which interact with COP1 to begin signal transduction. The UVR8 dimer develops through the formation of salt-bridges between individual UVR8 proteins. In this study the details of the dimerization are dissected, showing that several salt-bridge amino acids are necessary for the multiple functions of both the UVR8 dimer and monomer. Interestingly the authors show that UVR8 with conservative mutations of Asp96 and Asp107 to Asn96 and Asn107 are unable to form dimers yet retain wildtype responses to UV-B. This shows that monomeric UVR8 has the ability to normally initiate a signal transduction pathway and complicates our understanding of the in vivo role of the UVR8 dimer.

Karen Halliday (Edinburgh) is the corresponding author on this investigation into the broader impact of Arabidopsis phytochromes on carbon allocation and biomass production. Even though phytochrome mutants have reduced CO2 uptake they overaccumulate resources into sucrose and starch and show altered day:night growth rates. Overall this leads to reduced growth coincident with reduced expression of CELLULOSE SYNTHASE-LIKE genes. The authors demonstrate that phytochromes play a significant role in the control of biomass allocation and that they additionally differentially respond to external stresses. Evolutionarily this indicates that modification of phytochrome expression might be an important mechanism for responding to changing environments.

Simon Turner (Manchester) and Piers Hemsley (James Hutton Institute, University of Dundee) lead this research which amalgamates the work from their individual labs and assesses the role of S-acylation on the activity of cellulose synthase complex (CSC). They show that core subunits of the CSC, cellulose synthase A (CESA) proteins, require s-acylation for their localisation to the plasma membrane, which is necessary for their in vivo activity. The authors estimate that a CSC might contain over 100 S-acyl groups, which could significantly alter its hydrophobicity and its interactions within the membrane environment.

CES localisation: Taken from http://science.sciencemag.org/content/353/6295/166.full.pdf+html

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